Traditional anti-tumor therapy has employed cytotoxic agents, such as cis-platin, vinblastine, daunorubicin, and doxorubicin, to inhibit tumor cell growth. Recently, agents that inhibit the growth of new blood vessels have been found to be effective anti-tumor agents when used alone or in combination with chemotherapy (see, for example, Nature, 394:297 (1998).
Inhibition of the growth of new blood vessels around a tumor can inhibit tumor growth because, in solid tumors, there is a need for an extensive vascular network to support tumor growth and metastasis. Solid tumors cannot grow beyond the size of a pinhead (1 to 2 cubic millimeters) without inducing the formation of new blood vessels to supply the nutritional needs of the tumor. When the blood supply is limited, tumor growth and metastasis is suppressed.
At a critical point in the growth of a tumor, the tumor sends out signals to the nearby endothelial cells to activate new blood vessel growth. Two endothelial growth factors, VEGF and basic fibroblast growth factor (bFGF), are expressed by many tumors and seem to be important in sustaining tumor growth.
Angiogenesis is also related to metastasis. It is generally true that tumors with higher densities of blood vessels are more likely to metastasize and are correlated with poorer clinical outcomes. Also, the shedding of cells from the primary tumor begins only after the tumor has a full network of blood vessels. In addition, both angiogenesis and metastasis require matrix metalloproteinases, enzymes that break down the surrounding tissue (the extracellular matrix), during blood vessel and tumor invasion.
Several anti-angiogenesis drugs function by targeting specific molecules involved in new blood vessel formation. For others, the exact mechanism of action is unknown, but the compounds have been shown to be anti-angiogenic by specific laboratory tests (in the test tube or in animals).
About 15 proteins are known to activate endothelial cell growth and movement, including angiogenin, epidermal growth factor, estrogen, fibroblast growth factors (acidic and basic), interleukin 8, prostaglandin E.sub.1 and E.sub.2, tumor necrosis factor-.alpha., vascular endothelial growth factor (VEGF), and granulocyte colony-stimulating factor. However, most evidence points to a special role for VEGF (vascular endothelial growth factor). VEGF is a protein that is secreted from blood-deprived tissues and from some types of malignant cells.
VEGF appears to be important for tumor-induced vasculogenesis and enhanced vascular permeability. VEGF regulates angiogenesis by binding to specific receptors on nearby blood vessels, causing new vessels to form. Anti-VEGF agents have been proposed for inhibiting tumor growth.
One class of compounds which appears to be active against VEGF are anti-VEGF antibodies, including recombinant humanized monoclonal antibodies and chimeric antibodies. Sigma Chemicals commercially sells an anti-VEGF antibody (Human, clone no. 26503.11). Genentech's anti-VEGF and Agouron's AF3340 are also being investigated in various clinical trials. These antibodies purportedly have the ability to prevent VEGF from binding to its receptors.
Chimeric antibodies are antibodies in which an entire, intact, variable domain of a human antibody is substituted with that of a non-human one. In contrast, a humanized antibody includes only substitution of the six antigen-binding loops (CDRs) of the human antibody with those from a non-human one. Exchanging some non-CDR residues may also be required to attain binding of the humanized antibody that is similar to those of the parent non-human antibody.
Other known inhibitors of angiogenesis include angiostatin, endostatin, interferons, interleukin 1 (.alpha. and .beta.), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2. (TIMP-1 and -2). Small molecules, such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, have also been investigated for their ability to inhibit tumor anglogenesis.
Genetic material, such as antisense oligonucleotides and aptamers which inhibit VEGF have also been developed. Numerous articles have been published regarding oligonucleotide aptamers which purportedly have anti-VEGF activity. However, major limitations associated with the use of aptamers is that the body has numerous mechanisms for destroying exogenous DNA, such as exo and endonucleases. Further, there are synthetic difficulties associated with preparing useful amounts of oligonucleotides such as the anti-VEGF aptamers. Still further, the ability of these compounds to bind useful sites in vivo has yet to be demonstrated in a human model, in part due to the difficulty in preparing the compounds in a scale sufficient for clinical studies, and also in part due to the inability to develop useful animal models, which is related to the purported high affinity of the compounds for the human target sites.
The differences between standard chemotherapy and anti-angiogenesis therapy result from the fact that angiogenesis inhibitors target dividing endothelial cells rather than tumor cells. Anti-angiogenic drugs are not likely to cause bone marrow suppression, gastrointestinal symptoms, or hair loss--symptoms characteristic of standard chemotherapy treatments. Also, since anti-angiogenic drugs may not necessarily kill tumors, but rather hold them in check indefinitely, the endpoint of early clinical trials may be different than for standard therapies. Rather than looking only for tumor response, it may be appropriate to evaluate increases in survival and/or time to disease progression.
Drug resistance is a major problem with chemotherapy agents. This is because most cancer cells are genetically unstable, are more prone to mutations and are therefore likely to produce drug resistant cells. Since angiogenic drugs target normal endothelial cells which are not genetically unstable, drug resistance may not develop. Finally, anti-angiogenic therapy may prove usefll in combination with therapy directly aimed at tumor cells. Because each therapy is aimed at a different cellular target, it is possible that the combination will prove more effective than either therapy alone. For these reasons, development of new anti-angiogenic agents remains an important goal for cancer research.
Adenosine is known to be released in hypoxia. Numerous studies have shown adenosine to protect cells in the heart from ischemic damage. Adenosine binds to different receptor sites in the body. Four types of receptors have been identified, including the A.sub.1, A.sub.2a, A.sub.2b, and A.sub.3 receptors.
Adenosine has been shown to have protective roles in numerous animal models and in man (Am. J. Cardiol. 79(12A):44-48 (1997). For example, in the heart, both the A.sub.1 and A.sub.3 receptors offer protection against ischemia (Am. J. Physiol., 273(42)H501-505 (1997). However, it is the A.sub.3 receptor that offers sustained protection against ischemia (PNAS 95:6995-6999 (1998). The ability of adenosine to protect tumor cells against hypoxia has not been recognized by others.
The A.sub.3 receptor is believed to mediate processes of inflammation, hypotension, and mast cell degranulation, and apparently also has a role in the central nervous system. The A.sub.3 selective agonist IB-MECA induces behavioral depression and upon chronic administration protects against cerebral ischemia. A.sub.3 selective agonists at high concentrations were also found to induce apoptosis in HL-60 human leukemia cells. These and other findings have made the A.sub.3 receptor a promising therapeutic target.
Selective antagonists for the A.sub.3 receptor have been proposed for use as antiinflammatory and antiischemic agents in the brain. Recently, A.sub.3 antagonists have been under development as antiasthnatic, antidepressant, antiarrhythmic, renal protective, antiparlinson and cognitive enhancing drugs.
Recent studies in myocytes have shown the adenosine A.sub.3 receptors to be responsible for long term protection against ischemia (Liang and Jacobson, PNAS, 95:6995-6999 (1998)). While the present inventors have hypothesized that adenosine plays a protective role in other cell types, including tumor cells, in addition to myocytes, no efforts have been made to limit the protective effect of adenosine on tumor cells.
It is therefore an object of the present invention to provide compositions and methods of treatment of tumor cells which involve minimizing or eliminating the protective effect of adenosine on tumor cells.